Regulation of Cellular Zinc Homeostasis

细胞锌稳态的调节

• 批准号: 7862866
• 负责人: David J Eide
• 金额: $ 17.59万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-16 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862866
• 关键词: Binding; Biological Assay; Carrier Proteins; Cell physiology; Cells; Complex; Cytosol; Endoplasmic Reticulum; Environment; Eukaryota; Eukaryotic Cell; Fingers; Fluorescence Resonance Energy Transfer; Foundations; Golgi Apparatus; Growth; Health; Homeostasis; Human; Life; Mammalian Cell; Mammals; Mediating; Metabolism; Metallothionein; Metals; Mitochondria; Modeling; Molecular; Nutrient; Organelles; Organism; Oxidative Stress; Pathway interactions; Physiology; Play; Process; Proteins; Role; Saccharomyces cerevisiae; Source; System; Testing; Yeasts; Zinc; Zinc Fingers; Zinc deficiency; base; cell growth regulation; cofactor; extracellular; in vivo; public health relevance; response; sensor; uptake; zinc-binding protein

DESCRIPTION (provided by applicant): Zinc is an essential nutrient because of the important roles this metal plays as a catalytic and structural cofactor. Several zinc-dependent proteins reside within organelles such as the endoplasmic reticulum (ER), Golgi, and mitochondria. Therefore, transporter proteins are needed to distribute zinc into intracellular compartments. Because excess zinc can be toxic, cells also require homeostatic mechanisms to control the intracellular levels of free or labile zinc in the cytosol and within organelles. Cellular zinc homeostasis is achieved by several mechanisms including the control of zinc uptake, efflux, vesicular storage, and binding by metallothionein. In this proposal, another important facet of zinc homeostasis is considered, i.e. the control of zinc levels within intracellular organelles. These processes will be studied using the yeast Saccharomyces cerevisiae as a model eukaryotic cell. Preliminary results have raised five central hypotheses that provide the foundation for the specific aims of this proposal: 1) The yeast Msc2 and Zrg17 proteins form a heteromeric complex that transports zinc into the ER. In Aim 1, the composition of this complex will be assessed and its zinc transport activity will be characterized. 2) It is proposed that zinc finger domains will make useful probes of zinc status in the ER and other compartments of living cells. In Aim 2, new in vivo zinc sensors will be developed based on fluorescence resonance energy transfer (FRET). These and other assays to be developed will be used to define the role of Msc2/Zrg17 and other transporters in maintaining ER zinc homeostasis. 3) Zinc transport into the ER via the Msc2/Zrg17 complex is regulated in response to zinc status by both transcriptional and post-translational control mechanisms. In Aim 3, the molecular mechanisms of these regulatory systems will be dissected and their roles in maintaining ER zinc status determined. 4) It is proposed that zinc transport into the ER is mediated by transporters in addition to the Msc2/Zrg17 complex. In Aim 4, other zinc transporters for this compartment will be identified. 5) Several studies of mammalian cells showed that zinc deficiency leads to increased oxidative stress, the source of which has long been a mystery. In Aim 5, the hypothesis that the oxidative stress of zinc deficiency arises from the loss of ER zinc homeostasis will be tested. These aims represent a cohesive and comprehensive analysis of zinc homeostasis in the secretory pathway of eukaryotic cells.7. PUBLIC HEALTH RELEVANCE: The processes of intracellular zinc transport and homeostasis within organelles are essential for basic cellular function, physiology, and human health. Despite this importance, however, we know little about the transporters involved and how they are regulated in response to zinc status and other factors. As a result of our proposed studies, we will obtain a fundamental understanding of zinc metabolism in the secretory pathway of all eukaryotes and generate new probes of intracellular zinc that will be useful in our studies as well as in the analysis of zinc metabolism in mammals and other organisms.

描述（由申请人提供）：锌是一种必需的营养素，因为这种金属作为催化和结构辅因子发挥着重要作用。几种锌依赖蛋白存在于细胞器如内质网（ER）、高尔基体和线粒体内。因此，需要转运蛋白将锌分配到细胞内区室中。因为过量的锌可能是有毒的，细胞还需要稳态机制来控制细胞溶质和细胞器中游离或不稳定锌的细胞内水平。细胞锌稳态是通过几种机制实现的，包括锌的摄取、流出、囊泡储存和金属硫蛋白的结合。在这个建议中，锌稳态的另一个重要方面被认为是，即细胞内细胞器内锌水平的控制。这些过程将使用酵母酿酒酵母作为模型真核细胞进行研究。初步结果提出了五个中心假设，为本提案的具体目标提供了基础：1）酵母MSC 2和Zrg 17蛋白形成一种异聚复合物，将锌转运到ER中。在目标1中，将评估该复合物的组成，并表征其锌转运活性。2)有人建议，锌指结构域将成为有用的探针锌状态的ER和其他车厢的活细胞。目标2：基于荧光共振能量转移（FRET）技术开发新型体内锌离子传感器。这些和其他待开发的检测方法将用于确定Msc 2/Zrg 17和其他转运蛋白在维持ER锌稳态中的作用。3)锌转运到ER通过Msc 2/Zrg 17复合物调节锌状态的转录和翻译后控制机制。在目标3中，这些调节系统的分子机制将被解剖，并确定它们在维持ER锌状态中的作用。4)有人提出，除了Msc 2/Zrg 17复合物之外，锌向ER的转运还由转运蛋白介导。在目标4中，将确定该隔室的其他锌转运蛋白。5)对哺乳动物细胞的几项研究表明，缺锌会导致氧化应激增加，其来源长期以来一直是个谜。在目标5中，将测试锌缺乏的氧化应激来自ER锌稳态的丧失的假设。这些目标代表了真核细胞分泌途径中锌稳态的连贯性和全面性分析。公共卫生相关性：细胞内锌的转运和细胞器内的稳态过程对于基本的细胞功能、生理学和人类健康是必不可少的。然而，尽管如此重要，我们对所涉及的转运蛋白以及它们如何响应锌状态和其他因素进行调节知之甚少。由于我们提出的研究，我们将获得一个基本的了解锌代谢的分泌途径的所有真核生物，并产生新的探针的细胞内锌，这将是有用的，在我们的研究以及在哺乳动物和其他生物体的锌代谢的分析。